Crotamine in Crotalus durissus: distribution according to subspecies and geographic origin, in captivity or nature

Abstract Background: Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods: The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results: The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. Conclusion: These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.(AU)

Anti-Trimeresurus albolabris venom IgY antibodies: preparation, purification and neutralization efficacy

Snakebite incidence in southwestern China is mainly attributed to one of the several venomous snakes found in the country, the white-lipped green pit viper Trimeresurus albolabris. Since antivenom produced from horses may cause numerous clinical side effects, the present study was conducted aiming to develop an alternative antivenom antibody (immunoglobulin Y - IgY) from leghorn chickens. Methods IgY in egg yolk from white leghorn chicken previously injected with T. albolabris venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot, and its neutralizing assay was conducted on mice. Results Chickens injected multiple times with T. albolabris venom elicited strong antibody responses, and from their egg yolk IgY was isolated and purified, which exhibited a single protein band on SDS-PAGE and two bands (about 65 and 35 kDa, respectively) under reduced conditions. Immunoblot analysis revealed that these IgY are polyclonal antibodies since they bind with most venom components. In the neutralizing assay, all mice survived while the ratios of IgY/venom reached up to 3.79 (50.0 mg/13.2 mg). Conclusions IgY antibody response was successfully conducted in white leghorn chicken injected with T. albolabrisvenom. IgY against T. albolabris venom was obtained for the first time, and it exhibited strong neutralizing potency on mice. These results may lay a foundation for the development of IgY antivenom with clinical applications in the future.

Estudio clínico-epidemiológico de alteraciones hemorrágicas y necróticas causadas por el veneno de la serpiente de cascabel (crotalus durissus cumanensis) en pacientes venezolanos / Clinical and epidemiological study of hemorrhagic and necrotic alterations caused by the rattlesnake (crotalus durissus cumanensis) venom in venezuelan patients

Se describieron los efectos hemorrágicos, necróticos y edematosos de 135 pacientes provenientes de los estados Miranda, Aragua, Vargas y Distrito Capital, Venezuela, ocasionados por la mordedura de la serpiente cascabel común venezolana (Crotalus durissus cumanensis), durante los años 1998-2008. Los trastornos hemorrágicos, que tradicionalmente eran casi imperceptibles en los Crotalus venezolanos, hemos encontrado que hay evidencias francas de manifestaciones clínicas como: afibrinogenemia, alargamiento del tiempo de coagulación manual (TCM), tiempo parcial de tromboplastina (TTP) y tiempo de protrombina (TP), lo cual indica la presencia de estas fracciones hemorrágicas en el veneno de cascabeles nacionales. Se apreciaron diferencias entre ambos sexos, siendo predominante en el sexo masculino (82%). Sin embargo ha habido un aumento de incidencia significativa en el sexo femenino (17%). Por grupo etario, se observó predominancia entre 11 a 30 años de edad, en ambos sexos. El sitio de mordedura mayormente afectado fue el miembro superior (58,5%), con un porcentaje no menos significativo de miembros inferiores (40,7%). Estos hallazgos, permiten sugerir que el veneno de algunas serpientes cascabeles comunes en Venezuela, poseen un efecto sistémico sobre el músculo esquelético, y también efectos sobre capilares que generan edema, fenómenos hemorrágicos y necrosis, que habían pasado desapercibidos.

The bleeding, necrotic and edematous Snake bite effects from 135 patients of Miranda, Aragua, Vargas States and Capital District (Venezuela), caused by the Venezuelan common rattlesnake (Crotalus durissus cumanensis) from 1998 to 2008 were described. In bleeding disorders, which traditionally were almost imperceptible in Venezuelan Crotalus, we have found reliable evidence of clinical manifestations such as: afibrinogenemia, lengthening of the manual time of coagulation (MTC), and Partial Time of Thromboplastin (PTT) and Prothrombin time (PT), which indicates the presence of hemorrhagic fractions in the Venezuelan rattlesnake’s venoms. There were differences between the sexes, still predominant in male (82%). However, there has been an increase of significant impact on female (17%). By age, there was prevalence between 11 and 30 years old, both male and female. The mostly affected bite si te was upper limb (58,5%), with a no less significant percentage of lower limbs (40,7%). These findings, allowed us to suggest that some rattlesnake venoms have a systemic effect on skeletal muscle, and also effects on capillaries that generate swelling, hemorrhagic phenomena and necrosis.

Biochemical characterization and some biological properties of the phosphodiesterase I purified from Agistrodon bilineatus venom.

The venom phosphodiesterase I (PDE-I, EC 3.1.4.1) is useful in the elucidation of the structure and nucleotide sequence of nucleic acids. In the present study, PDE-I was purified from Agistrodon bilineatus venom by preparative native-PAGE. A single protein band was observed in analytical native-PAGE. The enzyme also gave a single band in SDS-PAGE with a molecular mass of 140 kDa. The position of the band was not altered in the presence of β-mercaptoethanol, suggesting the protein did not contain subunits. The enzyme was free from 5’-nucleotidase and alkaline phosphatase activities. It showed a broad optimum pH range (9.0-11.0), whereas the optimum temperature was found to be 600C, with activity decreasing at >650C. Energy of activation (Ea) was calculated to be 0.31. The PDE-I was a glycoprotein having 14% of carbohydrate content. The Vmax, Km, Kcat and Ksp values of the enzyme were 3.85 μM/min/mg, 8.3 × 10-3 M, 23s-1 and 46.4 M-1 Min-1 respectively. Cysteine caused a non-competitive inhibition with a Ki 6.3 × 10−3 M (IC50 of 1.6 mM), whereas ADP caused a competitive inhibition having Ki 0.8 × 10−3 M (IC50 5.4 mM). Glutathione, o-phenanthroline, zinc and EDTA inhibited the enzyme activity, whereas Mg2+ slightly potentiated the activity. The enzyme hydrolyzed thymidine 5’-monophosphate p-nitro-phenyl ester most readily (10-fold), while 3’-5’-cAMP was least readily hydrolyzed substrate. The enzyme up to 4.0 mg/Kg i.p was not lethal in mice. It exhibited an anticoagulant effect, and increased the normal clotting time of normal citrated human plasma, whereas the crude venom showed strong coagulant effect. The above results showed that the A. bilineatus PDE-I was very similar to that isolated from other snake venoms. The purification procedure described here is simple, rapid and reproducible and may prove useful to isolate pure protein for investigation into the contribution of this enzyme to the biological activities of A. bilineatus venom and PDE-I insight, in general.

Age-related biological activity of south american rattlesnake (Crotalus durissus terrificus) venom

An in vitro and in vivo comparative study was performed on the effects of Crotalus durissus terrificus venoms from a mother and its 15 newborns. The venoms were tested for protein content, lethality, proteolytic, myotoxic, hemorrhagic, and phospholipase A2 activity. The minimum coagulant dose in plasma and human fibrinogen, protrhombin, and Factor II activations were analyzed. The venoms were also analyzed by polyacrylamide gel electrophoresis (PAGE). This showed that despite similar total protein content, the biological effects of the venoms were different. Venom from young snakes exhibited higher enzymatic and coagulant activities and higher myotoxicity compared to the mother's. In addition, the PLA2 content paralleled myotoxicity. However, no difference could be detected in their toxicity (LD50 0.08 mg/Kg). High incidence of blood coagulation disorders and elevated circulating myoglobin may characterize systemic envenoming by young C. d. terrificus.

Clonagem, identificaçäo e análise de genes de peptídios tóxicos da cascavel sul americana, Crotalus durissus terrificus. Implicações evolutivas e funcionais / Cloning South American rattlesnake Crotalus durissus terrificus toxin peptides genes analysis and identification. Evolutions and functional implications

O veneno de animais contém um arsenal de toxinas que desencadeia respostas fisiológicas e bioquímicas específicas. A crotamina, um peptídio catiônico (4,4 kDa, pI 9,5), é um dos componentes mais abundantes do veneno de cascavel Sul Americana (Crotalus durissus terrificus). No Brasil, há populações de C. d. terrificus que expressam ou näo a crotamina no veneno. Em um único espécime de C. d. terrificus crotamina-positivo, foram isolados cDNAs precursores de duas isoformas de crotamina, dentre as quais a crotamina Ile-19, presente somente no veneno de C. d. ruruima. Análise por Northern blot de RNA total e mensageiro de glâdulas de C. d. terrificus crotamina-positivo e -negativo, indica que a expressäo...

Intraspecific variation in the venom electrophoretic profile of recently captured Crotalus durissus terrificus (Laurenti, 1768) snakes

The aim of this study was to compare individual venom samples of Crotalus durissus terrificus recently captured in the wild to evaluate possible differences in venom protein composition. Protein levels were quantified by biochemical method (Biuret) and then submitted to electrophoresis. Electrophoresis studies of native protein were performed in vertical slabs of polyacrylamide gel (PAGE), in an alkaline discontinuous buffer system, with a concentration of 10 per cent in the separation gel. SDS-PAGE was performed in PhastGel (8-25). Both gels were stained with Coomassie Blue. Gels were analyzed using the VDS-Pharmacia device. Our results indicate that all analyzed venom samples showed different protein composition, although common protein fractions were detected in some individual samples. Differences were observed between the different individual venom samples and so in the same specimen in relation to the time of collection, for both techniques used. Diet did not influence the variability of venom composition. There is a significant difference in native venom protein composition of males and females.

A report on a case of melanism in a specimen of Crotalus durissus terrificus (Laurenti, 1768)

This report documents a case of a melanic specimen of Crotalus durissus terrificus (Laurenti, 1768) found in Bofete, Säo Paulo State, Brazil. The authors describe this melanic snake, determine the electrophoretic pattern of its venom, and compare the venom of this specimen against that of normal Crotalus durissus terrificus. This report is very important because melanism is a rare chromatic anomaly.

Purification and partial characterization of an l-amino acid oxidase from bushmaster snake (Surucucu pico de jaca) Lachesis muta venom

L-amino-acid oxidase(L-AO) form the venom of Lachesi muta muta was purified 72 times (38%) by gel filtration on Sephadex G-100, followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephacryl S-300. The protein was shown to be homogeneous by polyacrylamide gel electroforesis, immunoelectrophoresis, immunodiffusion and isoelectric focusing. Its specific activity was 44.4 units/mg protein, using 7.5 mM L-leucine as substrate a nd O-dianisidine as electron donor,at pH 7.6 and 25§C. The increase in absorbance at 436 nm was recorded. The enzyme was characterized as a glycoprotein with an S20,w=6.72,MW=138,000 and pI=5.2. It presented maxima at 389 and 460 nm and contained 2 mol of FAD per mole protein

Characterization of a monoclonal antibody that neutralizes the hyaluronidase activity of Russell's viper venom.

Three monoclonal antibodies (WPN1, WPN2 and WPN3) raised against a partially purified fraction of Russell's viper venom (RVV) were characterized. All three monoclonal antibodies reacted with crude RVV when tested by ELISA, but only two (WPN1, WPN2) neutralized its hyaluronidase activity. WPN1 was the more potent and was effective at an antigen: antibody ratio of 1:3. Furthermore, WPN1 was shown to recognize only the 14,000 MW component of crude RVV. This has been identified in a previous study to be hyaluronidase. This antibody was also found to recognize some components of Calloselasma rhodostoma venom which also possesses potent hyaluronidase activity. The potential therapeutic role of antibodies that neutralize the hyaluronidase component of snake venoms should be investigated further.

South American snake venom proteins antigenically related to bothrops asper myotoxins

1. the presence of proteins antigenically related to Bothrops asper myotoxins in various snake venoms, mainly from South America, was investigated by using poluclonal and monoclonal antibodies. 2. Myotoxin-like components were detected in the bothrops venoms from South america, and in the venoms of Crotalus atrox (North america), Trimerusurus flavoviridis (Japan), and Micrurus alleni (Costa Rica). 3. Cross-reactive components detected in several Bothrops venoms show a common subunit of 15-16 LDa by sodium dodcyl sulphate-polyacrylamide gel electrophoresis, although significant charge variations are evident by immunoelectrophoresis. 4. It is concluded that proteins antigenically related to B. asper nyotoxins are relatively common in the genus Bothrops and, in the light of findings discussed, are likely to posses myotoxic activity

Model studies of crotamine self-association

1. The self-association of crotamine in the concentration range from 10 to 40 microg/ml was studied at pH 6, 25-C, and at low ionic strength by monitoring the effect of protein concentration on the absorbance at 196 nm. 2. Of the several mathematical models tested, an equilibrium between monomers and trimers with an association constant of 1.50 x 10 M-2 gives an adequate representation of the phenomenon. However, a non-ideal, two-stage model describing an equilibrium among monomers, dimers, and trimers gives the best fit of the experimental data. 3. The equilibrium constants found for dimerization and trimerization were 1.70 x 10 M-1 and 3.37 x10 M-1, respectively. 4. This model was confirmed by polyacrylamide gel electrophoresis where a trimer band was separated from an interconverting monomer-dimer band

Anticoagulant effect of myotoxic phospholipase A2 isolated from the venom of the snake Bothrops asper (Viperidae)

La fosfolipasa A2 miotoxica del veneno de Bothrops asper (tercipelo) prolonga el tiempo de recalcificación de plasmas de cinco especies de mamíferos. La fosfolipasa A2 hidroliza los fosfolípidos del plasma, en tanto que su acción inhibitoria es revertida cuando se adicionan fosfolípidos plaquetarios. Estas dos observaciones sugieren que la acción anticoagulante se debe a una alteración de los fosfolípidos plasmáticos necesarios para la coagulación. Tanto la actividad enzimática como la anticoagulante se mantuvieron aún después de calentamiento a 95è-C durante 10 min. Pese a su acción anticoagulante in vitro, la fosfolipasa A2 no prolonga el tiempo de coagulación luego de inoculación intravenosa en ratones

Fraccionamento do veneno Crotalus durissus terrificus por cromatografia de exclusäo molecular / Fractitionation of Crotalus durissus terrificus venom by molecular exclusion chromatography

Através da técnica de cromatografia de exclusäo molecular utilizando Sephadex G-75 foram estudadas as diferentes fraçöes do veneno de Crotalus durissus terrificus, uma das serpentes peçonhentas mais comuns no Brasil. Foram obtidos 4 picos (correspondentes às fraçöes 32,60,86 e 103) com peso molecular variando de 4.000 a 150.000. As fraçöes de todo o diagrama de gel filtraçäo foram triadas através de reaçäo de imunoeletroforese a fim de se verificar suas cargas e velocidade de migraçäo. As linhas de precipitaçäo encontradas foram comparadas às 11 linhas apresentadas pela reaçäo de imunoeletroforese do veneno total contra o soro anti-crotálico. Constatou-se que as fraçöes de um mesmo pico apresentavam características próprias com exeçäo da frraçäo 54 (subida do pico II) que mostrou diferenças significativas em relaçäo à fraçäo 60. Após a triagem foram escolhidas as fraçöes de cada pico onde as linhas de precipitaçäo foram mais nítidas e intensas, para estudo de identidade através da reaçäo de difusäo radial dupla e letalidade comparada a concentraçäo de 0,0625 mg/ml do veneno total que corresponde a DL50 em camundongo pelo método de SPEARM & KARBER. As fraçöes 32, 86 e 103 correspondentes respectivamente aos picos I, III e IV apresentaram letalidade nula ou negligenciada e a fraçäo II foi a mais tóxica